Trehalose catalytic shift is an intrinsic factor in Mycobacterium tuberculosis that enhances phenotypic heterogeneity and multidrug resistance

Drug-resistance (DR) in many bacterial pathogens often arises from the repetitive formation of drug-tolerant bacilli, known as persisters. However, it is unclear whether Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis (TB), undergoes a similar phenotypic transition. Recent metabolomics studies have identified that a change in trehalose metabolism is necessary for Mtb to develop persisters and plays a crucial role in metabolic networks of DR-TB strains. The present study used Mtb mutants lacking the trehalose catalytic shift and showed that the mutants exhibited a significantly lower frequency of the emergence of DR mutants compared to wildtype, due to reduced persister formation. The trehalose catalytic shift enables Mtb persisters to survive under bactericidal antibiotics by increasing metabolic heterogeneity and drug tolerance, ultimately leading to development of DR. Intriguingly, rifampicin (RIF)-resistant bacilli exhibit cross-resistance to a second antibiotic, due to a high trehalose catalytic shift activity. This phenomenon explains how the development of multidrug resistance (MDR) is facilitated by the acquisition of RIF resistance. In this context, the heightened risk of MDR-TB in the lineage 4 HN878 W-Beijing strain can be attributed to its greater trehalose catalytic shift. Genetic and pharmacological inactivation of the trehalose catalytic shift significantly reduced persister formation, subsequently decreasing the incidence of MDR-TB in HN878 W-Beijing strain. Collectively, the trehalose catalytic shift serves as an intrinsic factor of Mtb responsible for persister formation, cross-resistance to multiple antibiotics, and the emergence of MDR-TB. This study aids in the discovery of new TB therapeutics by targeting the trehalose catalytic shift of Mtb.


Introduction
The World Health Organization (WHO) estimated that between 2000 and 2020, over 200 million people were ill with tuberculosis (TB), and more than 35 million died from the disease. 1 In 2023, one fourth of world's population was still infected with Mycobacterium tuberculosis (Mtb), the bacterium that causes TB.Conventional TB treatment typically involves the administration of multiple antibiotics over a prolonged period, sometimes extending up to two years.This extended duration is largely due to the wide range of antibiotic tolerance exhibited by Mtb bacilli within the infected host. 2,35][6][7] Persisters are largely resistant to antibiotic effects and can typically only be eliminated through a combination of multiple antibiotics over an extended treatment period.The lengthy treatment duration is a major extrinsic factor that contributes to noncompliance among TB patients, which, in turn, leads to the emergence of multidrug-resistant (MDR) TB.The uncontrolled emergence and spread of MDR-TB pose signi cant challenge in the global TB pandemic.To combat MDR-TB, it is crucial to understand the intrinsic factors within Mtb that contribute to the development of MDR and to discover new treatments that speci cally prevent the emergence of MDR-TB during chemotherapy.
Persisters are a phenotypic variant that is transiently but highly tolerant to nearly all TB antibiotics.Their formation is determined by metabolic remodeling rather than genetic changes. 8The formation of persisters and the accompanying antibiotic tolerance are well-known intrinsic factors that Mtb employs to survive antibiotic effects.Intriguingly, the level of antibiotic susceptibility remains unaltered when persisters regrow under antibiotic-free conditions.0][11] Additionally, persisters are recognized as a source of genetic mutationmediated MDR.[14][15] Intensive research has focused on elucidating the molecular mechanisms behind persister biology in an effort to advance TB chemotherapeutics.7][18] Separate studies have revealed bacteria-speci c intrinsic factors that bacteria use to adapt to these environmental stresses.One example is the toxin-antitoxin (TA) modules of Escherichia coli. 19,20Overexpression of the toxin or downregulation of the antitoxin signi cantly reduces growth rates and induces levels of antibiotic tolerance, a phenomenon frequently observed in the persister-rich subfraction of E. coli.2][23][24] Recent metabolomics studies have shown that Mtb phenotypic heterogeneity is attributed to the formation of a new subpopulation with altered central carbon metabolism (CCM) activity.This remodeling includes the capacity to co-catalyze multiple carbon sources, such as glycolytic and gluconeogenic carbons, to support Mtb's full replication rate.It also involves the catabolic remodeling of cell wall glycolipids to enhance Mtb persister biology, bypassing the oxidative branch of the TCA cycle to downregulate the production of NADH.Additionally, Mtb can reciprocally induce carbon ux through the glyoxylate shunt or methylcitrate cycle and enhance the biosynthesis of succinate, followed by active secretion, to optimize membrane bioenergetics under diverse environmental stresses. 8,13,22,25Thus, metabolic remodeling serves as a crucial intrinsic factor employed by Mtb persisters used to survive antibiotic effects.[28][29][30][31] Phenotypic heterogeneity has recently garnered signi cant attention as an additional intrinsic factor of Mtb that promotes adaptive evolution. 32Expanding the bacterial subpopulation with a diverse range of metabolic activities can lead to the emergence of genotypes distinct from those of the original population.The newly generated subpopulation can serve as a source of bacilli that withstand bactericidal stresses.Recent studies have utilized mathematical simulation to model drug resistance during bacterial infection.These studies have revealed that even small increases in mutation rates considerately accelerate the frequency of emerging drug resistance, primarily due to induced phenotypic heterogeneity. 33,34A proof-of-concept study in yeast supported these ndings by demonstrating that initial drug-resistance mutations in a small fraction resulted in higher minimal inhibitory concentration (MIC) of antibiotics and accelerated the emergence of MDR.Additionally, increased expression of e ux pumps heightened mutation rates through a previously unknown mechanism involving mutS and further increased phenotypic heterogeneity. 35,36An intriguing example of how metabolic network remodeling promotes phenotypic heterogeneity is the random emergence of persisters, often referred to as a "gambler" subpopulation. 33When bacterial pathogens are exposed to antibiotics, only a small fraction of the bacilli activate their DNA repair systems. 37Although the SOS stringent response is triggered, only a speci c subset of bacilli experiences increased levels of ROS and the subsequent activation of the stress response.Since both the SOS and general stress responses are essential for mutagenic DNA break repair, persisters exhibit a signi cantly higher mutagenic rate.Furthermore, ample evidence has shown that adaptive metabolic network remodeling plays a crucial role in enhancing phenotypic heterogeneity, thereby providing a source of gaining evolutionary advantages through frequent DNA mutagenesis.
We have recently identi ed that trehalose synthase (TreS) plays a crucial role as a mediator in driving metabolic remodeling and phenotypic heterogeneity, leading to the formation of Mtb persisters capable of surviving antibiotic effects. 13,22Trehalose, a non-reducing glucose disaccharide, is highly abundant in Mtb.It serves both as a carbohydrate store and a core component of cell wall glycolipids, such as trehalose monomycolate (TMM) and trehalose dimycolate (TDM). 38Metabolomics studies conducted with Mtb bacilli collected from in vitro bio lm cultures revealed that a TreS-centered catalytic redirection is critical to Mtb persister biology.This redirection channels free trehalose towards the biosynthesis of intermediates in the CCM, providing an alternate source of energy and antioxidants, while diverting it away from the biosynthesis of TMM and TDM.As anticipated, a treS-de cient Mtb mutant (ΔtreS), which lacks the trehalose catalytic shift, exhibited heightened sensitivity to all tested TB antibiotics.These ndings underscore the importance of the TreS-mediated trehalose catalytic shift and subsequent metabolic remodeling in Mtb persister formation and enhanced antibiotic tolerance.We have also recently developed TreS-speci c inhibitors and con rmed their potential as adjunctive therapeutic candidates. 39Notably, the trehalose catalytic shift appears to be more active in DR-TB clinical isolates than in drug-sensitive (DS)-TB clinical isolates.This suggests that the trehalose catalytic shift may be a strategy employed to facilitate the emergence of drug resistance.
In this study, we demonstrated that inactivating the trehalose catalytic shift using the CRISPRi-dCas9 technology not only limited persister formation and phenotypic heterogeneity but also reduced rates of drug resistance against key rst-line TB antibiotics, rifampicin (RIF) and isoniazid (INH).We employed mathematical modeling to show that mycobacterial bacilli enhance their chances of acquiring drug resistance by increasing trehalose catalytic shift activity and phenotypic heterogeneity.The mathematical modeling also indicated that the conversion frequency from persisters to drug-resistant mutants was nearly the same for both wildtype and ΔtreS, suggesting that the metabolic remodeling involved in inducing persister formation also plays a signi cant role in the emergence of drug resistance.Additionally, we identi ed new Mtb subfraction, termed pre-resistant bacilli, which appears before the acquisition of drug resistance.Unlike persisters, these bacilli can grow even under antibiotic stress, and their formation is largely attributed to enhanced trehalose catalytic shift activity.This phenotypic heterogeneity, resulting from the formation of persisters or pre-resistant bacilli, is a critical intrinsic factor in Mtb that contributes to the accumulation of drug resistance.Therefore, the trehalose catalytic shift represents a potential target for adjunctive therapeutic strategies, not only to better understand Mtb phenotypic heterogeneity but also to prevent the emergence of MDR-TB cases.

Results
Recent reports indicate that trehalose acts as a growth-permissive carbon source for DR-TB clinical isolates. 13However, the trehalose-mediated growth was reversed when co-treated with a TreS-speci c inhibitor, validamycin A (ValA). 13 Metabolomics pro ling further supported the central role of the TreScentered trehalose catalytic shift in the metabolic networks of DR-TB clinical isolates.The catalytic conversion of trehalose into intermediates of glycolysis and the pentose phosphate pathway (PPP) suggests that DR-TB clinical isolates prefer trehalose as a substrate for the biosynthesis of CCM intermediates, rather than for the production of cell wall glycolipids like TDM.These observations led us to hypothesize that the trehalose catalytic shift not only contributes to transient antibiotic tolerance but also plays a critical role in the emergence of multidrug-resistant mutants.
Trehalose metabolism differs in DR-TB and DS-TB clinical isolates.
To further investigate the trehalose metabolism networks in DR-TB and DS-TB clinical isolates, we collected a total of 75 TB clinical isolates from the TB clinical isolate library at the International Tuberculosis Research Center (ITRC).This collection included 15 DS-TB, 15 rifampicin single-resistant (RSR)-TB, 15 MDR-TB, 15 extensively drug-resistant (XDR)-TB, and 15 totally drug-resistant (TDR)-TB clinical isolates (Table S1).All strains in each category were cultured in Middlebrook 7H9 liquid medium (m7H9) supplemented with sodium butyrate (SB), a permissive carbon source for all clinical isolates. 13,23e growth of DS-TB and DR-TB clinical isolates was enhanced by the addition of 20 mM trehalose; however, the growth of DR-TB, but not DS-TB, clinical isolates was reduced when co-treated with ValA (Fig. S1A).Although the heterogenous growth kinetics of DR-TB clinical isolates complicated appropriate statistical analyses, the speci c impact of ValA on the growth of DR-TB clinical isolates in m7H9 containing trehalose clearly indicated that DR-TB clinical isolates rely more on the TreS activity to utilize exogenously supplied trehalose compared to DS-TB clinical isolates (Fig. S1A).To examine their metabolic networks, we extracted the total metabolome after culturing the isolates in m7H9 containing 20 mM trehalose.We determined the trehalose-induced metabolic networks of TB clinical isolates by monitoring approximately 200 TB metabolites and comparing their abundances in DR-TB clinical isolates with those in DS-TB clinical isolates.Using bioinformatics tools available in MetaboAnalyst (v.6.0),we identi ed metabolic networks unique to DR-TB clinical isolates by speci cally focusing on those involved in trehalose consumption.Hierarchical clustering analysis revealed distinct metabolomics patterns between all DR-TB and DS-TB clinical isolates as depicted in the heatmap and phylogenetic tree (Fig. S1B).Principal Component Analysis (PCA) further con rmed the divergence in metabolomics patterns between the two categories of TB clinical isolates (Fig. 1A).These analyses demonstrated that the metabolic networks in DS-TB clinical isolates used to consume trehalose differed from those in MDRand XDR-TB clinical isolates but were relatively similar to those of RSR-TB clinical isolates (Figs.1A,   S1B).We identi ed metabolites in DR-TB clinical isolates that were signi cantly altered compared to those in DS-TB clinical isolates, conducted pathway mapping, and found that trehalose metabolism, the D-alanine pathway, and the PPP were among the top-ranked pathways.Targeted metabolomics analysis indicated that trehalose abundance was signi cantly greater in all DR-TB clinical isolates (Fig. 1B, left panel).Furthermore, the biosynthesis of glycolysis and PPP intermediates, such as glucose 6-phosphate (Glc6P), pentose 5-phosphate (Pen5P), and sedoheptulose 7-phosphate (S7P) in DR-TB clinical isolates was either similar to or signi cantly greater than that in DS-TB clinical isolates (Fig. 1B, middle panels).This suggests that a substantial portion of exogenously supplied trehalose is utilized for the biosynthesis of intermediates in glycolysis and PPP in DR-TB clinical isolates. 13Consistent with previous ndings, 23 the level of phosphoenolpyruvate (PEP), the most downstream intermediate in glycolysis was similar across all clinical isolates (Fig. 1B, right panel).In contrast to the metabolites in upper glycolysis and PPP, those in the TCA cycle of all DR-TB clinical isolates were either unaltered or downregulated (Fig. S1C).Notably, the treS mRNA expression in all DR-TB clinical isolates remained unaltered, although it was slightly induced in TDR-TB clinical isolates (Fig. 1C).Collectively, these ndings indicate that the metabolic networks involved in trehalose consumption are organized differently between DS-TB and DR-TB clinical isolates, with regulation occurring independently of transcriptional changes.
TreS-de cient M. smegmatis phenocopied Mtb mutants that lack trehalose catalytic shift.
To study the role of trehalose catalytic shift in the development of drug resistance in mycobacterial bacilli, we employed the recently developed CRISPRi-dCas9 technique to inducibly deplete treS gene expression in M. smegmatis (Fig. S2A). 40,41The CRISPRi treS mutants of M. smegmatis (termed ItreS SM ) were cultured in the mid-log phase, and treS knockdown was induced using various concentrations of anhydrotetracycline (ATc).The e cacy of treS mRNA knockdown was assessed by qRT-PCR with treatment at 200 ng/mL ATc resulting in approximately 90% suppression (Fig. 2B, left panel).We also created IotsA SM to knockdown otsA, a gene responsible for encoding trehalose 6P synthase involved in Mtb trehalose metabolism, but not associated with the trehalose catalytic shift.Similar to the observation in treS-de cient Mtb (ΔtreS), 13 ItreS SM produced persister-like bacilli within the in vitro bio lm culture (referred to as bio lm-persisters) at a signi cantly lower level than wildtype following ATc treatment.In contrast, both IotsA SM and wildtype were able to form mature bio lm-persisters, despite showing no discernible growth defects in Sauton media (Fig. S2C, D).ItreS SM without ATc was included as a complement condition and exhibited the ability to form bio lm-persisters at a level similar to that of wildtype (Fig. S2D).Targeted metabolomics of ItreS SM revealed that the inability to form intact bio lmpersisters was primarily due to impaired trehalose catalytic shift, which affected the trehalose-mediated carbon ux through glycolysis and the PPP (e.g., Glc6P, glyceraldehyde 3P, and S7P) (Fig. S2E).Recently, the depletion of both PEP abundance and the PEP/pyruvate ratio has been identi ed as a metabolic strategy employed by Mtb to induce persister formation, slow its replication rate, and enhance antibiotic tolerance. 23Notably, ItreS SM showed accumulated PEP as compared to that of wildtype (Fig. S2E).As a result, ItreS SM exhibited increased susceptibility to antibiotics, such as RIF, INH, and BDQ compared to wildtype or IotsA SM (Fig. S2F), similar to the phenotype observed in ΔtreS Mtb. 13 These ndings collectively indicate that ItreS SM phenocopies ΔtreS Mtb.
The trehalose catalytic shift is an adaptive strategy to emerge drug-resistant mycobacterial mutants.
If Mtb persisters survive antibiotic-induced bactericidal oxidative stresses, such as ROS which are known DNA mutagen, their prolonged survival may be linked to the development of drug-resistant mutations.The metabolic strategies employed by Mtb persisters during this stage are directly or indirectly involved in the emergence of drug resistance. 42,43To examine whether the trehalose catalytic shift is a strategy functionally associated with the emergence of drug-resistant mutants, we employed a classical Luria-Delbrück uctuation assay to determine the rates of emerging spontaneous drug-resistant mutants in both wildtype and ItreS SM . 44,45We found that the drug-resistance rates of wildtype against RIF ranged from 5.1 X 10 − 7 to 1 X 10 − 6 mutations per generation (Fig. 2A, left panel).The drug-resistance rates of ItreS SM without ATc were comparable to those of wildtype.RIF-resistant colonies were con rmed by spotting them on m7H10 containing high concentrations of RIF, up to 100 µg/mL (Figs.2B and S3A).The uctuation assay and the spot assay indicated that the mean rate of RIF resistance in wildtype was approximately 6.6-fold greater than in ItreS SM .Additionally, we determined the INH-resistance rates of wildtype, which ranged from 1.1 X 10 − 5 to 5.5 X 10 − 6 mutations per generation, while the rates for ItreS SM ranged from 1.8 X 10 − 6 to 1.0 X 10 − 6 mutations per generation.The wildtype exhibited INH resistance development at levels approximately 5.4-fold greater than that of ItreS SM (Fig. 2A, right panel).
These ndings suggest a functional link between the trehalose catalytic shift and the frequency of drug resistance development in mycobacterial bacilli against rst-line TB antibiotics, irrespective of the modes-of-action.
We also performed a co-culture competition assay using wildtype expressing green uorescent protein (GFP) and ItreS SM expressing red uorescent protein (RFP) (Fig. S3B).With these two strains, we measured relative viability following cyclic exposure to bactericidal concentrations of RIF or Dcycloserine (DCS), with intermittent washing with antibiotic-free PBS, and established G1 to G5 subcultures (Fig. S3B).Flow cytometry analysis was utilized to determine the relative abundance of wildtype::GFP and ItreS SM ::RFP within the G0 to G5 subcultures (Fig. 2C).The iterative cycle of treatment with RIF or DCS, followed by regrowth in antibiotic-free m7H9, led to a gradual accumulation of wildtype bacilli within the subcultures.In the G4 and G5 subcultures, GFP intensity became saturated but never reached 100% (Fig. 2C).This nding indicates that the G4 and G5 subcultures may contain drug-resistant bacilli from both wildtype and ItreS SM .Indeed, the spot assay showed that G3 subculture was the rst generation to exhibit the drug-resistant phenotype, and the lag phase period during the regrowth kinetics of the G4 and G5 subcultures was nearly identical to that of naïve bacilli (Fig. S3C, D).These ndings indicate that the trehalose catalytic shift represents an intrinsic strategy of Mtb that is functionally associated with the tness cost required for natural selection and a regrowth advantage in the face of intermittent antibiotic stresses.To further support these ndings, we conducted a uctuation assay using M. smegmatis overexpressing treS (pTreS) and found that the extracopy of treS conferred mycobacterial bacilli resistance to RIF at levels approximately 2.0-fold greater than those of wildtype (Fig. S3E).
DR mutants are metabolically heterogenous by forming bacilli harboring greater trehalose catalytic shift activity.
Using the uctuation assay and RIF spot assay, we isolated 10 RIF-resistant M. smegmatis colonies, designating them as Flux RIF #1-#10 (Figs.2A, B, and S3A).Consistent with the growth kinetics of previously reported DR-TB clinical isolates, 13 the growth patterns of all Flux RIF and naïve bacilli were nearly identical in antibiotic-free m7H9 (Fig. S4A).However, while naïve bacilli were unable to form colonies on m7H10 containing RIF concentrations of 25 µg/mL or higher, all Flux RIF bacilli successfully grew on the plates (Figs.2B and S3A).Notably, Flux RIF #1 and #2 bacilli carried an L 452 P mutation in the RIF-resistance determining region (RRDR), 46 a mutation well-known to be associated with RIF resistance in many DR-TB clinical isolates. 47,48In contrast, Flux RIF #3-#10 developed RIF resistance without any mutations in the RRDR region.To investigate the role of the trehalose catalytic shift in the observed drugresistant phenotype of the Flux RIF bacilli, we monitored their growth kinetics after supplementing with 20 mM trehalose.The addition of trehalose enhanced the growth rates of both groups of bacilli.Since ValA has minimal impact on M. smegmatis TreS activity, we employed the CRISPRi-dCas9 technique to deactivate treS in the Flux RIF bacilli.We found that suppression of treS partially hindered the trehaloseinduced growth of Flux RIF bacilli, whereas it had little effect on the growth of naïve bacilli (Fig. S4A).This suggests a more pronounced TreS-centered trehalose catalytic shift activity in Flux RIF bacilli compared to naïve bacilli.Our conclusion was further supported by metabolomics pro ling, which revealed that the levels of Glc6P, fructose 1,6-bisphosphate (FBP), and S7P were signi cantly higher in Flux RIF bacilli than in naïve bacilli, even though both strains exhibited similar levels of trehalose abundance (Fig. 3A).In contrast to the glycolysis and PPP intermediates, there were no noticeable changes in the levels of TCA cycle intermediates (Fig. S4B).Therefore, we conclude that the catalytic activities responsible for utilizing exogenous trehalose to biosynthesize glycolysis and PPP intermediates are considerably higher in Flux RIF bacilli, consistent with observations from DR-TB clinical isolates (Fig. 1B). 13In addition, we observed that Flux RIF bacilli maintained high levels of PEP despite their antibiotic tolerance, likely because they continued to replicate even in the presence of RIF (Figs. 3A and S3D). 23Consistent with the metabolomics pro le and drug-resistant phenotype, Flux RIF bacilli exhibited higher expression levels of treS mRNA compared to naïve bacilli, with expression levels particularly elevated in the RRDR mutationfree Flux RIF #3-#10 bacilli (Figs.3B and S4C).Moreover, Flux RIF bacilli contained a larger subfraction with lower membrane potential (ΔΨm) and lower ATP levels than naïve bacilli, whose bioenergetic states resembled those of Mtb persisters (Fig. 3C, D). 13,23 As a result, RIF antibiotic penetration into Flux RIF bacilli occurred at signi cantly reduced levels compared to naïve bacilli, a nding further supported by the EtBr permeability assay (Fig. 3E).Taken together, these observations indicate that Flux RIF bacilli exhibit increased metabolic heterogeneity by expanding the population with a greater trehalose catalytic shift and lower bioenergetic activities.This metabolic heterogeneity may contribute to the initiation of persister formation, antibiotic tolerance, and the development of drug resistance.
The trehalose catalytic shift confers mycobacterial cells with greater metabolic heterogeneity.
0][51] Recent studies have demonstrated that DR-TB clinical isolates exhibit lower TDM abundance in their cell wall due to increased trehalose catalytic shift activity. 13,23,52To de ne a functional connection between the trehalose catalytic shift of Flux RIF bacilli and their ability to enhance metabolic heterogeneity, we utilized previously reported Red Molecular Rotor-trehalose (RMR-tre), a uorogenic dye that speci cally labels mycobacterial cell wall glycolipids containing trehalose as a carbohydrate core, such as TDM. 535][56] We quanti ed the intensity of RMR-tre labeling using FACS before and after treatment with sublethal doses of RIF.As expected, the RMR-tre labeling pattern of naïve bacilli was relatively homogenous before antibiotic treatment.However, it became heterogenous after RIF treatment, as evidenced by an increase in the subfraction of RMR-tre high bacilli.This phenomenon likely occurs because mycobacterial bacilli with induced trehalose catalytic shift activity preferentially consume preexisting trehalose as a substrate for CCM intermediates, resulting in a greater level of RMR-tre incorporation compared to endogenous trehalose.Notably, these RMR-tre high bacilli were absent in ΔtreS Mtb (Fig. S5A).To further investigate the extent to which the trehalose catalytic shift contributes to the formation of the RMR-tre high subfraction and the associated metabolic heterogeneity, we repeated the RMR-tre labeling assay using pTreS SM , M. smegmatis overexpressing treS, and ItreS SM .Our observations revealed that the RMR-tre high subfraction substantially overlapped with that of pTreS SM , whereas it was absent in ItreS SM , similar to what was observed in ΔtreS Mtb (Figs. 4A-C, S5A).This nding underscores the functional essentiality of the trehalose catalytic shift in promoting metabolic heterogeneity in response to bactericidal antibiotics.Interestingly, the fraction of RMR-tre high bacilli was signi cantly larger in Flux RIF bacilli compared to naïve DS-bacilli, even prior to antibiotic treatment (Figs. 4D and S5B, C).To determine whether the RMR-tre high subfraction in Flux RIF bacilli primarily consists of a viable population following treatment with bactericidal antibiotics, we tracked changes in the abundance of RMR-tre high and RMR-tre low subfractions after exposure to bactericidal doses of RIF.We observed a profound decrease in the RMR-tre low subfraction, with the RMRtre high subfraction becoming predominant (Fig. S5D).This suggests that the metabolic heterogeneity induced by the formation of the RMR-tre high subfraction is largely attributed to an enhanced trehalose catalytic shift, which is functionally related to antibiotic tolerance and the accumulation of drug-resistant mutations.Further intriguingly, this phenomenon was more pronounced in Flux RIF #3-#10 bacilli than in Flux RIF #1 and #2 bacilli.Flux RIF #1 and #2 bacilli, which harbored the L 452 P mutation in the RRDR, maintained the RMR-tre low subfraction as a dominant population even after antibiotic treatment, although there was a slight reduction in its abundance (Fig. S5D).This may occur because Flux RIF #3-#10 bacilli exhibit RIF resistance due to a larger fraction harboring high trehalose catalytic shift activity.
In contrast, the RIF resistance in Flux RIF #1 and #2 bacilli is likely mediated by mutations in the RIF target gene.Treatment with RIF rendered all Flux RIF #3-#10 bacilli relatively more homogenous, either by inducing the trehalose catalytic shift in the RMR-tre low subfraction or by speci cally killing the less drugtolerant RMR-tre low subfraction (Fig. S5D).This indicates that RMR-tre high bacilli may represent a signi cant source of viable bacilli following treatment with bactericidal antibiotics.Overall, the trehalose catalytic shift is an intrinsic factor of Mtb that elevates metabolic heterogeneity and enhances its ability to survive longer under antibiotic pressure by generating the RMR-tre high subfraction, which readily contributes to the formation of persisters and pre-resistant bacilli.
The trehalose catalytic shift is necessary to elevate drug resistance frequency by increasing the persister subfraction.
Pathogenic bacteria can transiently acquire a drug-tolerant phenotype through a non-genetic mechanism by forming persisters.They subsequently regrow as a species when the effects of antibiotics diminish.This cycle repeats until drug-resistant mutants emerge (Fig. 5A).The phenotypic reversibility between drug-sensitive bacilli and drug-tolerant persisters occurs when antibiotic priming is intermittent.
Continuous antibiotic pressure, however, leads to the accumulation of drug-resistant mutations. 57,58 have employed mathematical modeling to create analytical formulas that predict the impact of a trehalose-catalytic shift on the kinetics of reversibility and the observed clone-to-clone uctuations within the population that survives antibiotic stresses.This surviving population ultimately serves as a reservoir for drug-resistant bacilli (Fig. 5A). 59,60To capture the emergence of drug-tolerant persisters during population growth, we have developed a model in which single bacilli reversibly switch between drug-sensitive and drug-tolerant states. 61Once a bacillus becomes drug-tolerant, it remains in that state for multiple generations before reverting to a drug-sensitive state (Fig. 5A).Our previous work has modeled such a reversible switching in the context of a uctuation assay, allowing us to analytically predict the expected statistical variation in the number of drug-tolerant bacilli across colonies derived from a single bacillus.Our analysis of the uctuation assay data using this reversible switching model indicates that ItreS SM persisters are more unstable than wildtype persisters, reverting to a drug-sensitive state more quickly.Additionally, our ndings reveals that the observed lower number of resistant colonies in the ItreS SM compared to wildtype (Fig. 5B) is predominantly due to a six-fold lower rate of persister formation in ItreS SM (see Mathematical Modeling in the Method section).3][64][65][66] Drugresistant mutants exhibit metabolic similarities to Mtb persisters (Figs. 3, 4, S4, and S5).Thus, we conclude that mycobacterial bacilli evolve into drug-resistant mutants through the repetitive formation of drug-tolerant persisters and pre-resistant bacilli.The trehalose catalytic shift serves as a strategy to enhance the subfraction of persisters and pre-resistant bacilli under high levels of ROS damage, thereby facilitating the emergence of drug-resistant mutants.
RIF-resistant mycobacterial cells are also resistant to INH and BDQ.
Reports from TB clinical isolates at Taiwan Medical Center indicate that 94.6% of RIF-resistant strains were also resistant to INH while only 0.5% were mono-resistant to RIF. 67 A similar pattern was observed in the retrospective TB case studies conducted in New York City between 2010 and 2021. 68These ndings suggest that RIF resistance may serve as a predictive biomarker for MDR-TB.Therefore, we hypothesized that RIF-resistant strains could possess a metabolic advantage that confers greater tolerance to second antibiotics, such as INH, even without prior exposure to these antibiotics.RMR-tre labeling patterns indicate that Flux RIF bacilli contain a high abundance of RMR-tre high subfraction (Figs.4D and S5B, C).To investigate this hypothesis, we conducted a minimum inhibitory concentration (MIC) shift assay using selected Flux RIF bacilli and their CRISPRi treS mutant, referred to as ItreS Flux , comparing their antibiotic sensitivity to that of naïve bacilli.Flux RIF bacilli exhibited signi cantly higher tolerance to INH, with MIC values of approximately 3.82 µg/mL, compared to around 1.84 µg/mL for naïve bacilli.However, this INH tolerance diminished in ItreS Flux after treatment with ATc, resulting in an MIC value at around 1.49 µg/mL (Fig. 6A, left panel).This nding was not observed in ItreS Flux without treatment with ATc.This suggests that Flux RIF bacilli are better equipped to withstand the effects of INH, likely due to their greater abundance of the RMR-tre high subfraction (Figs.4D and S5D).A spot assay performed on m7H10 containing bactericidal doses of INH corroborated the results of the MIC shift assay (Figs.6B and S7A).Additionally, Flux RIF bacilli demonstrated higher tolerance to BDQ as well, underscoring the signi cant role of the trehalose catalytic shift in cross-resistance to various TB antibiotics (Fig. 6A, right panel).This is further supported by the fact that the ITRC TB clinical isolate library includes only 15 RSR-TB clinical isolates (less than 1%) among a collection of over 1,500 clinical isolates.Surprisingly, the inverse relationship of cross-resistance was not clearly detected.INH-resistant bacilli (referred to as Flux INH ), obtained from the uctional assay (Fig. 2A, right panel), were collected and tested for their antibiotic sensitivity against RIF.The MIC shift assay and colony size measurement conducted on two randomly selected Flux INH bacilli revealed that they were signi cantly more sensitive to RIF compared to naïve bacilli (Fig. 6C, D), likely due to their increased RIF permeability (Fig. S7B, upper panel).This altered membrane permeability was further supported by the EtBr permeability assay (Fig. S7B, lower panel).INH is a prodrug that requires structural activation through the formation of an NAD + adduct to exhibit its antimicrobial activity. 69As shown in Fig. 3, Flux RIF bacilli demonstrated distinct metabolic networks compared to naïve bacilli, primarily attributed to a higher trehalose catalytic shift and concurrently lower membrane bioenergetics, characterized by reduced levels of NAD + , ΔΨm, and ATP (Fig. 3C, D).This metabolic state likely in uences the formation of INH-NAD adducts.The increased cross-resistance of Flux RIF bacilli to INH or BDQ was signi cantly downregulated by inhibiting treS using CRISPRi-dCas9 technique (Fig. 6A).This supports the hypothesis that the trehalose catalytic shift contributes to the emergence of MDR-TB cases.
The trehalose catalytic shift enables HN878 W-Beijing strain to acquire a high frequency of multidrug resistance.
Clinical Mtb strains are categorized into phylogeographic lineages 1 through 7, each exhibiting varying capacities for acquiring MDR mutations. 45,70Lineage 2 strains, including the HN878 W-Beijing strain (HN878), have been associated with a heightened risk of MDR-TB emergence on a global scale.Our ndings suggest that the trehalose catalytic shift in Mtb contributes to an increased frequency of MDR mutations by promoting the formation of persisters and cross-resistance to multiple antibiotics (Figs. 4, 5, and 6), Therefore, we hypothesize that elevated trehalose catalytic shift activity in HN878 plays a key role in its propensity to accumulate MDR mutations more frequently than other lineage strains.To investigate this hypothesis, we examined TreS activity in HN878 following exposure to sublethal doses of RIF.The expression of treS mRNA in HN878, as well as in lineage 4 strains such as Erdman or CDC1551, was notably upregulated in response to RIF treatment.Interestingly, the induction of treS mRNA in HN878 increased by approximately 7.3-fold compared to the untreated controls, which was signi cantly higher than the 2 to 3-fold increase observed in lineage 4 strains (Fig. 7A).We also found that HN878 exhibited faster growth rates than the lineage 4 strains in m7H9 containing trehalose as the sole carbon source (Fig. 7B).Co-treatment with ValA restored trehalose-mediated growth to levels comparable to those of lineage 4 strains, suggesting that trehalose may serve as a more favorable carbon source for HN878, likely due to its higher TreS activity (Fig. 7B).The catalytic activities involved in converting consumed trehalose into glycolysis and PPP intermediates such as Glc6P, Pen5P, and S7P, were signi cantly higher in HN878 than in lineage 4 strains, further supporting our hypothesis (Fig. 7C).
Collectively, these ndings suggest that HN878 undergoes a greater trehalose catalytic shift compared to lineage 4 strains, leading to the development of MDR mutations more frequently in HN878 than in other lineage strains.
To further validate the functional importance of the trehalose catalytic shift in HN878 for the emergence of drug-resistant mutants, we conducted a uctuation assay using HN878 and lineage 4 strains, both with and without ValA, as well as the CRISPRi treS mutant of HN878 (ItreS HN ), CDC1551 (ItreS CDC ), or Erdman (ItreS Erd ) (Figs. S2A and S8A).Consistent with previous literature, 45 we observed that HN878 exhibited approximately a 5.0-fold higher frequency of developing RIF resistance compared to lineage 4 strains (Fig. 7D, left panel).Treatment with ValA signi cantly reduced the mutation rates to levels comparable to those of lineage 4 strains (Fig. 7D, left panel).Similar results were observed with the CRISPRi treS mutants, where the rates of drug-resistant mutations for ItreS HN and ItreS CDC against RIF became comparable (Fig. 7D, right panel).Furthermore, HN878 showed a signi cantly higher MIC of RIF (~ 0.06 µg/mL) due to its enhanced trehalose catalytic shift activity, compared to lineage 4 strains (~ 0.03 µg/mL).However, when co-treated with ValA or in ItreS HN , the MIC value decreased to approximately 0.02 µg/mL (Figs.7E and S8B, C).To establish a link between the enhanced trehalose catalytic shift and its metabolic heterogeneity, as well as persister formation and drug tolerance in HN878, we utilized the most probable number (MPN) assay.This recently innovated method monitors the abundance of total persisters, which includes traditional persisters and differentially detectable (DD) bacilli under RIF treatment and nutrient-starved conditions. 71,72We found that the frequency of persister formation in HN878 was the highest among all clinical strains tested in this study (Fig. S8D).The reduction rate after co-treatment with ValA (Fig. S8D, left panel) or using ItreS HN (Fig. S8D, right panel) was the largest, suggesting that the high frequency of MDR development in HN878 is largely attributed to its greater trehalose catalytic shift activity and the resulting persister formation.According to our mathematical modeling results (Fig. 5), the frequent emergence of MDR-TB cases linked to infections with HN878 is primarily due to elevated levels its trehalose catalytic shift activity and persister formation.Thus, the trehalose catalytic shift represents a promising target for novel adjunctive therapeutics aimed at preventing the emergence of MDR-TB.

Discussion
Persister formation is a widespread adaptive strategy among bacterial pathogens including Mtb, allowing them to survive the effects of antibiotics for extended periods without developing genetic mutations that confer drug resistance. 8,73The pathogenic cycle of TB includes an intractable latent infection stage, during which Mtb bacilli often enter a persister state.In this state, they can opportunistically recur, increasing the bacterial burden and serving as a source for potential genetic mutations that lead drug resistance. 33,74,75Compared to heritable drug resistance, the biology of Mtb persisters is still in the early stages of investigation.The present study indicates that mycobacterial persisters are indeed an adaptive method that plays a crucial role in the optimal pathogenic cycle of TB.1][82] Notably, the accumulation of ROS resulting from antibiotic effects has been identi ed as a primary factor that kills invading bacilli.However, when target pathogens survive, ROS can induce DNA mutagenesis. 30,83,84Thus, prolonged survival following a persister state, accompanied by regrowth through metabolic remodeling strategies, is directly associated with the development of populations harboring genetic mutation-mediated drug resistance. 12,85These metabolic adaptive strategies represent a mechanism underlying the accelerated emergence of MDR-TB.
A handful of investigations into the key metabolic remodeling strategies required for persister formation have begun.Our metabolomics studies, utilizing Mtb persisters collected from in vitro bio lm cultures or under hypoxic stress, have validated the functional importance of preexisting Mtb cell wall glycolipids as an alternative source for carbon nutrients. 13,22Bioinformatic analysis of the metabolomics data revealed that trehalose metabolism was one of the most signi cantly altered pathways compared to replicating Mtb.These ndings suggest that Mtb persisters shift the catalytic direction of trehalose metabolism to biosynthesize intermediates in glycolysis and the PPP, a strategy termed the trehalose catalytic shift.Indeed, the ΔtreS of Mtb, which lacks this catalytic shift activity, exhibited hypersensitivity to rst-line TB antibiotics such as INH and RIF.Trehalose serves as a structural component of Mtb cell wall glycolipids, such as TDM, which plays a crucial role in immunomodulatory interactions with the host immune system.Separately, sulfolipid-1 (SL-1) also contains trehalose as a core carbohydrate, and its function has recently been reported to be linked to the opportunistic transmission of Mtb bacilli to healthy individuals. 86Therefore, the trehalose catalytic shift plays multiple roles, including carbon storage, essential components for persister biology, antibiotic tolerance, immune evasion, and transmission. 38,87is study has revealed an additional role of the trehalose catalytic shift in accelerating the development of permanent MDR in Mtb.The bacilli maintain viability by forming persisters through this shift activity, even under bactericidal levels of oxidative stress.This process induces DNA mutagenesis via the activation of the trehalose catalytic shift.Notably, RIF single-resistant Mtb bacilli tend to exhibit increased levels of antibiotic tolerance against a second antibiotic, even without prior exposure.
Consequently, this adaptive strategy can contribute to the emergence of MDR-TB.Furthermore, our study has shown that the trehalose catalytic shift enhances phenotypic heterogeneity.By inhibiting treS expression through CRISPRi or chemically deactivating TreS with ValA, a key enzyme in the trehalose catalytic shift, we observed a signi cant reduction in the emergence of DR mycobacterial mutants against clinically relevant TB antibiotics.Fascinatingly, our mathematical modeling has clari ed that the trehalose catalytic shift uniquely facilitates persister formation and provides phenotypic stability, preventing persisters from reverting to the DS-state.This suggests that the frequency of DR mutant emergence is predominantly in uenced by the extent of persister formation and its associated phenotypic heterogeneity and stability.We concluded this based upon our ndings that the transition rates from persisters to permanent DR mutants were nearly identical between wildtype and ΔtreS.Therefore, the metabolic remodeling strategies that promote Mtb persister formation represent a novel source of antibiotic targets aimed not only at eliminating Mtb persisters but also at averting the onset of MDR-TB.
We have developed a novel technique to monitor mycobacterial phenotypic heterogeneity resulting from the active trehalose catalytic shift by labeling with an RMR-tre uorogenic dye in conjunction with FACS analysis.In our recent report, 53 we demonstrated that RMR-tre serves as a substrate of Ag85, an enzyme involved in the biosynthesis of TDM, at levels comparable to free trehalose.In this study, we identi ed that RMR-tre labeling intensity was proportional to the TreS activity (Fig. A-C).This is likely because mycobacterial cells with higher TreS direct a larger fraction of trehalose toward the biosynthesis of CCM intermediates, resulting in limited internal trehalose availability for TDM biosynthesis.Consequently, when exogenous RMR-tre is supplied, mycobacterial cells with greater TreS utilize more RMR-tre as a substrate for TDM, whereas TreS-de cient mycobacterial cells rely more on endogenous trehalose.In response to antibiotic treatment, Mtb exhibited an accumulation of RMR-tre high bacilli, a subfraction displaying a labeling pattern similar to pTreS but absent in ItreS SM or ΔtreS Mtb.This suggests that the activation of the trehalose catalytic shift leads to increased metabolic and phenotypic heterogeneity in Mtb, resulting in a newly formed population that exhibits tolerance to antibiotic effects.The Flux RIF bacilli showed a higher abundance of the RMR-tre high subfraction compared to naïve DS-bacilli, underscoring the essential role of the trehalose catalytic shift in the metabolic networks of DR-bacilli and thus underlying mechanisms that contribute to their cross-resistance to other antibiotics.Treatment with bactericidal concentrations of antibiotics caused a notable increase in the proportion of bacilli exhibiting high TreS activity.This effect may arise from the greater antibiotic susceptibility of the subfraction with low TreS activity or the alteration of their metabolic heterogeneity, which promotes the induction of TreS activity.
The exploration of metabolic strategies beyond the trehalose catalytic shift is warranted, as a signi cant portion of ItreS SM or ΔtreS Mtb strains continue to develop drug-resistant mutations, albeit at a notably reduced rate.Consistent with the data obtained from Flux RIF bacilli, metabolomics analysis of DR-TB clinical isolates revealed distinctly different metabolic activities involved in trehalose catalysis compared to DS-TB clinical isolates.These DR-TB clinical isolates exhibited biochemical similarities to Mtb persisters, characterized by dysregulated membrane bioenergetics and active glycolysis and the PPP which serve as alternate sources of energy and antioxidants.Additionally, they showed a reduced abundance of cell wall TDM, a proin ammatory ligand of Mtb, as part of an immune evasion strategy. 13,22,88The catabolic remodeling of TDM provides infected Mtb bacilli with a spatiotemporal advantage, allowing them to maintain their latent status without depending host nutrients or provoking excessive immune system stimulation.TB clinical isolates utilize host fatty acids or cholesterol as primary carbon sources, which require endergonic metabolic pathways such as the TCA cycle, glyoxylate shunt, methylmalonyl CoA pathway, and methylcitrate cycle, followed by gluconeogenic reactions.Gluconeogenesis primarily involves endergonic reactions that consume energy to biosynthesize carbohydrate intermediates.Therefore, the trehalose catalytic shift represents a catalytic advantage, enabling the exploitation of metabolic networks largely composed of exergonic reactions to support the energy needs and antioxidants requirements essential for Mtb persister biology, antibiotic tolerance, and the eventual survival of MDR mutants.
Labeling Flux RIF bacilli with RMR-tre dye revealed that those carrying RRDR mutations exhibited signi cantly fewer Flux RIF bacilli compared to the RRDR mutation-free Flux RIF bacilli (Fig. S5B, C).
Additionally, treatment with bactericidal antibiotics selectively eliminated the RMR-tre low subfraction, a phenomenon that was more pronounced in the RRDR mutation-free Flux RIF bacilli (Fig. S5D).This nding suggests that Flux RIF bacilli lacking RRDR mutations may serve as a primary reservoir for the future development of DR mutations and the emergence of permanent MDR mutants.Consequently, these preresistant subfractions of bacilli may require a signi cantly higher level of trehalose catalytic shift to sustain their drug-resistant phenotype.Collectively, Mtb could achieve a permanent DR phenotype through the formation of Mtb persisters or pre-resistant bacilli by inducing the trehalose catalytic shift activity.Similar to carbapenem-resistant Enterobacteriaceae clinical isolates (CRE), 89 Mtb persisters may revert to a DS-state once the effects of antibiotics diminish (Fig. 5A).In contrast, pre-resistant bacilli were found to be phenotypically stable, consistently managing their metabolic networks with a high level of trehalose catalytic shift.The functional relevance of the trehalose catalytic shift in the formation of Mtb persisters and/or pre-resistant bacilli was con rmed by genetically inactivating treS in Flux RIF bacilli (Fig. 4D, E).
Consistent with previous ndings, we observed that the greater frequency of MDR mutations in lineage 2 clinical strains such as HN878 is largely attributed to their increased trehalose catalytic shift activity.This enhanced activity is linked to the induced formation of persisters and pre-resistant bacilli.Our mathematical modeling suggests that the greater frequency of persister formation is phenotypically associated with increased likelihood of enhancing phenotypic heterogeneity and the emergence of MDR-TB.The cross-resistance study revealed that Mtb bacilli with elevated levels of trehalose catalytic shift are more susceptible to develop MDR-TB mutations.This research illuminates the metabolic basis for the increased frequency of MDR-TB cases in HN878 infections.Targeting the trehalose catalytic shift in HN878 offers a novel therapeutic strategy to enhance the e cacy of clinically relevant TB antibiotics by preventing both the formation of persisters and the emergence of MDR-TB cases.We recently demonstrated that certain trehalose structural analogues can disrupt Mtb persister formation and antibiotic tolerance by inhibiting TreS-centered trehalose catalytic shift activity, thereby enhancing the antimicrobial effects of INH or RIF. 39The potential antimicrobial synergy of these compounds with clinically relevant TB antibiotics against HN878 infection warrants further investigation.

Materials & Methods
Bacterial Strains, Culture Conditions, and Chemicals.

Metabolite extraction and LC-MS analysis
M. smegmatisor Mtb-laden lters used for metabolomics pro ling were generated and incubated at 37°C for 5 days to reach the mid-log phase of growth. 21To prepare for lter-culture-based metabolomics pro ling, M. smegmatis or Mtb grown on agar-supported lters were treated with Trehalose and/or ValA to expose larger inocula.M. smegmatis or Mtb-laden lters were metabolically quenched by immersing them in a precooled mixture of acetonitrile:methanol:H 2 O (40:40:20, v:v:v) at -40°C.Metabolites were extracted using mechanical lysis with 0.1-mm zirconia beads in a Precellys tissue homogenizer for 4 min at 6000 rpm, repeated twice under continuous cooling at or below 2°C.The lysates were clari ed by centrifugation and then ltered through a 0.22-µm Spin-X column.The residual protein content of metabolite extracts was measured using a BCA protein assay kit (Thermo Scienti c) to normalize the samples to cell biomass.
Extracted metabolites were separated using a Cogent Diamond Hydride type C column (gradient 3) with a mobile phase consisting of solvent A (ddH 2 O with 0.2% formic acid) and solvent B (acetonitrile with 0.2% formic acid).The mass spectrometer utilized was the Agilent Accurate Mass 6230 time of ight (TOF), coupled with an Agilent 1290 liquid chromatography (LC) system.Dynamic mass axis calibration was achieved through continuous infusion of a reference mass solution using an isocratic pump with a 100:1 splitter.This con guration resulted in mass errors of 5 ppm and mass resolution ranging from 10,000 to 25,000 over the m/z of 62-966 atomic mass units, with a dynamic range of 5 log 10 .Detected ions were identi ed as metabolites based on unique accurate mass-retention time identi ers for masses displaying the expected distribution of accompanying isotopomers.Metabolites were quanti ed using a calibration curve generated from chemical standards spiked into a homologous Mtb extract (1:10 diluted with metabolite extraction solution) to account for matrix-associated ion suppression effects.The abundance of metabolites was analyzed with Agilent Qualitative Analysis B.07.00 and Pro nder B.07.00 software (Agilent Technologies), employing a mass tolerance of < 0.005 Da.Data analysis including clustered heatmap, hierarchical clustering, principal component analysis, volcano plot, and pathway enrichment analysis was conducted using MetaboAnalyst (ver.6.0).All data obtained from metabolomics pro ling represent the average of at least two independent triplicates.
qRT-PCR analysis M. smegmatis or Mtb strains were grown in m7H9 until they reached mid-log phase.Bacilli were harvested by adding an equal volume of guanidine thiocyanate buffer.Total RNA was extracted with TRIzol reagent and the Purelink RNA mini kit (Invitrogen), following the manufacturer's instructions.Genomic DNA was removed from the samples using the Turbo DNA-free kit (Invitrogen).cDNA was synthesized from 500 ng of RNA using the iScript Kit (Bio-Rad) and quantitative PCR was performed with a C1000 Thermal Cycler.Primers and probes were designed using the PrimerQuest™ Tool (Integrated DNA Technologies), as detailed in Table S2.Fold changes were calculated based on ΔΔCt values normalized to the transcript levels of the sigA housekeeping gene and presented as log 2 values.

CRISPRi knockdown generation
Single guide RNA (sgRNA) was designed to target the 3'-end of the non-template strand open reading frame of the target genes, consisting of a ~ 20 nucleotide sequence located 5' of an effective protospacer adjacent motif (PAM) sequence within the region.The PLJR962 M. smegmatis CRISPRi backbone plasmid was ampli ed in E. coli, selected with kanamycin (50 µg/mL), and then digested with BsmBI restriction enzymes (NEB) before being cleaned and puri ed.The designed oligo primers (see Table S2) were annealed and ligated into the BsmBI-digested plasmid backbone.Competent M. smegmatis cells were prepared by washing mid-log phase cultures multiple times in ice-cold 15% glycerol solution.The recombinant plasmid of interest was introduced into the competent cells via electroporation using a Pulse Controller II and Gene Pulser II (BioRad).Transformed liquid cultures were grown to mid-log phase and plated on m7H9 containing 50 µg/mL kanamycin to select for the recombinant colonies.These colonies were subsequently regrown in m7H9 containing kanamycin and incubated with ATc (at 200 ng/mL) for at least 1 day to induce target gene repression.The endogenous knockdown of the target genes of interest was con rmed by qRT-PCR.

Luria-Delbrück Fluctuation Assay and analysis
The original classical uctuation assay was modi ed for this 45 For the generation of single-cell suspension of M. smegmatis wildtype and ItreS SM , cultures were diluted to an OD 595 of 0.000005 in 200 µL within 96-well plates.The cultures were then regrown until they reach an OD 595 of 0.7-1.0.A total of 100 µL from 60 randomly selected colonies were plated on m7H10 containing either 100 µg/mL RIF or 200 µg/mL INH.Spontaneous RIF-or INH-resistant colonies were counted after incubating for up to 10 days.To determine the total viable input cell number, the remaining 100 µL of the cultures were serially diluted and plated on m7H10 without antibiotics.Mutation rates were calculated using Lea-Coulson method (m/Nt where m, number of resistant colonies; Nt, total input). 90-culture competition assay The plasmids pTE-OX-GCT5 and pGMEH-p38-mRFP (Addgene) were transformed into wildtype M.
smegmatis and ItreS SM to generate wildtype::GFP and ItreS SM ::RFP strains, respectively.Using these strains, we assessed the relative viability of wildtype and treS de cient strains after cyclic exposure to bactericidal concentrations of RIF or D-cycloserine (DCS).Brie y, equal volumes of mid-log phase cultures of wildtype::GFP and ItreS SM ::RFP, both at an OD 595 of ~ 0.7, were mixed to create G0 (untreated) culture.This culture was treated with 100 µg/mL RIF or DCS for 1 day, after which it was washed with PBS and resuspended in antibiotic-free m7H9.This resulted in a new culture at an OD 595 of 0.05, which was then re-grown until it reached an OD 595 of ~ 0.7, referred to as the G1 culture.The G1 culture was treated again with the same concentrations of RIF or DCS for 1 day, washed with PBS, resuspended in fresh m7H9, and re-grown until it reached an OD 595 of ~ 0.7 to create the G2 culture.This procedure was repeated until we obtained the G5 culture.Flow cytometry was employed to detect the relative abundance of wildtype and ItreS SM during the generation of G0 to G5 cultures.
RRDR sequencing of lab made RIF resistant M. smegmatis The rpoB gene sequences of the Flux RIF mutants were compared to the reference rpoB genes for wildtype M. smegmatis. 91ApE plasmid-editing software was utilized to identify mutations.Flux RIF strains were streaked onto LB agar plates and incubated at 37°C overnight to isolate colonies for sequencing.The RRDR (RIF Resistance Determining Region) of the rpoB gene was assessed using Sanger Sequencing (Quintara Biosciences) with the following primers: Msmeg-rpoB-fwd (5'gctgatccagaaccagatcc-3') and Msmeg-rpoB-rev (5'-gatgacaccggtcttgtcg-3').
Membrane -membrane potential, NAD/NADH, ATP For membrane potential (ΔΨm) measurement, cultures were grown in m7H9 to mid-log phase and concentrated to an OD 595 of ~ 1.0 in fresh m7H9.The cultures were treated with 15 µM DiOC 2 and incubated for 40 min at 37°C, followed by washing with PBS to remove any extracellular dye.As a positive control for membrane depolarization, one culture was treated with 5 µM of the protonophore carbonyl-cyanide 3-chlorophenylhydrazone (cccp) (Invitrogen), while PBS served as a vehicle control.The assay was conducted in black clear-bottom 96-well plates (Costar), and a SpectraMax M4 spectro uorimeter (Molecular Devices) was used to measure green uorescence (488 nm/530 nm) and shifts to red uorescence (488 nm/610 nm).ΔΨm was calculated as the ratio of red uorescence to green uorescence, with each condition measured in triplicate.
To assess intrabacterial ATP and NADH/NAD levels, M. smegmatis-laden lters generated for the metabolomics pro ling were used.Intrabacterial ATP concentrations were measured using the BacTiter Glo Microbial Cell Viability Assay kit, following the manufacturer's instructions (Promega).NAD and NADH concentrations were measured using the FluroNAD/NADH detection kit, also following the manufacturer's instructions (Cell Technology).Bacterial metabolism was rapidly quenched by plunging the lters into the rst solvent in each kit.
RMR-tre labeling Flow cytometry RMR-tre was synthesized as previously reported and characterized by nuclear magnetic resonance (NMR) spectroscopy. 53Wildtype M. smegmatis, ItreS SM , pTreS SM , Flux RIF , or ItreS Flux cultures in mid-log phase were treated with 1X MIC RIF for 1 day.The cultures were then stained with RMR-tre uorogenic dye at a nal concentration of 10 µM and incubated at 37°C for 1 hour.Following incubation, the cultures were sorted using a ow cytometer (Attune NxT, Thermo Fisher Scienti c) and the reported values represent the gated cell fraction.Data were exported from the ow cytometry cytometer and analyzed using FlowJo software (BD Biosciences).Error bars represents the standard deviation from biological replicates.

Mathematical modeling
The classical uctuation assay was modi ed (Fig. S6A).To generate single lineage bacilli of M.
smegmatis wildtype and ItreS SM , cultures were diluted to an OD 595 of 0.00005 in 200 µL and dispensed into 96-well plates.The cultures were regrown until they reach an OD 595 of 0.7-1.0.A total of µL from 60 randomly selected wells were plated on m7H10 containing 100 µg/mL RIF, and spontaneous RIF-resistant colonies were counted after incubating for 10 days.To determine the total viable input cell number, the remaining 100 µL of cultures were serially diluted and plated on antibiotic-free m7H10.To infer the reversible switching rates between drug-sensitive and drug-tolerant states, we utilized recent mathematical work on calculating these rates from the uctuation assay.The variability quanti ed by the coe cient of variation (standard deviation divided by the mean) -is given by: ( where is the time (normalized to the bacterial doubling time) for which single bacillus colonies are expanded, is the average time (also normalized to the bacterial doubling time) spent in the drugtolerant state, and is the fraction of bacilli that are persisters. 59,60on long-term antibiotic exposure, each drug-tolerant bacilli irreversibly transition to a permanent drugresistant state with a small probability .Conditioned on the number of drug-tolerant bacilli , the number of drug-resistant colonies will follow a binomial distribution.The clone-to-clone variation in is given by: ( where and denote the average and the coe cient of variation of , respectively.Using (2), measurements of the number of colonies growing on antibiotics across single bacillus lineages on can estimate : This estimate can then be used to estimate from (1).Analyzing the uctuation assay data on from 60 single-cell lineages, we estimate: for the wildtype genotype, where denoted the 95% con dence interval as estimated by bootstrapping.Using this estimate in (1) with (i.e., each single bacillus was expanded for 30 cell generations before plating on RIF-containing plates) and the average frequency of drug-tolerant persisters as , we obtain the transient heritability of wildtype persister state to be Chromatography system, as previously reported. 21,23,92The intrabacterial RIF concentration was calculated as [RIF] drug only -[RIF] ltrate .Three biological replicates were tested per group.
M. smegmatis wildtype and drug-resistant strains were grown in m7H9 until an OD 595 of 0.7 was reached.Final 5 mL cultures were centrifuged at 13,000 rpm for 3 min, after which the supernatant was discarded and the pellet was washed with PBS.The OD 595 was then adjusted to 0.4 with PBS, and glucose was added to a nal concentration of 0.4%.Ethidium bromide (EtBr) was added at a concentration of 8 mg/mL, and 100 µL aliquots were transferred to each well of black, clear-bottom 96well plates (Costar).A SpectraMax M5 spectro uorometer (Molecular Devices) was used to measure uorescence, with excitation and emission wavelength set to 530 nm and 595 nm, respectively.Fluorescence data were acquired every 60 sec for 60 min.
MPN (the most probable number) method to detect the total persister bacilli The MPN method was conducted as previously reported. 72Brie y, HN878, H37Rv, Erdman, and CDC1551 strains in mid-log phase were grown in m7H9, harvested by centrifugation at 5,000 rpm for 8 min, and washed twice with PBS.The cultures were then centrifuged at 800 rpm for 8 min to generate a single-cell suspension.The supernatant was transferred to a separate tube and diluted with PBS to achieve an OD 595 of 0.1.A CFU assay was used for the inoculum quanti cation.A total of 20 mL of the single-cell suspension was prepared for each condition, transferred to a vented ask, and incubated at 37°C for 2 weeks.The starved cultures were then split into two 8 mL cultures in vented asks and treated with either 100 µg/mL RIF with or without 200 µM Validamycin A (ValA).The asks were incubated at 37°C for 5 days.After incubation, cultures were harvested by centrifugation at 5,000 rpm for 8 min, washed with PBS, and resuspended in 1 mL PBS.A MPN method was then performed by diluting 15 µL of the 1 mL culture in 135 µL of m7H9 in 96-well plates.Ten-fold serial dilutions were carried out in the 96-well plates, which were stored at 37°C.The OD 595 was measured after 3 weeks and again 5 weeks.The remaining culture from the assay was used for a CFU assay, with dilutions between ranging from 10 − 5 and 10 − 1 .ItreS HN and ItreS CDC treated with or without ATc were also included to conduct the MPN assay.

Statistical analysis
All data were by Prism (v10.0;GraphPad Software).Signi cant differences were calculated by unpaired Student's t-test and one-or two-way ANOVA using multiple-comparison tests as speci ed or the nonparametric Mann-Whitney test for skewed data.P values less than 0.05 were considered statistically signi cant.

Declarations
Author L.J.J., E.S., C.S., B.M.S., and E.H. designed research studies; L.J.J., L.S.K., D.S., L.G., G.S., S.A., and P.J.O.conducted experiments; L.J.J., G.L., S.G., S.A., P.J.O., B.M.S., and E.H. analyzed data; R.J. provided materials; L.J.J., S.A., P.O.J., B.M.S., and E.H. wrote the manuscript.and ItreS SM expressing red uorescence protein (RFP) was subjected to intermittent exposure to RIF over a total of 5 cycles, referred to as G0-G5 cultures.The relative enrichment of wildtype and ItreS SM in G0 to G5 cultures was calculated via ow cytometry and is represented as a percentage.Gray bar represents ItreS SM ; black bar represents wildtype.drug-tolerant bacilli (red).In this model, mycobacterial bacilli can reversibly switch between drugsensitive and drug-tolerant states.Once a bacillus becomes drug-tolerant, it remains in that state for multiple generations before reverting to a drug-sensitive state.Following prolonged antibiotic exposure, each drug-tolerant bacillus irreversibly achieves permanent drug resistance.To assess the impact of the trehalose-catalytic shift on the frequency of each step in phenotypic transitioning, wildtype M. smegmatis and ItreS SM were utilized in a uctuation assay as depicted in Fig. S6A.(B) The rates forming drug-resistant mutants in wildtype and ItreS SM against RIF were calculated using the classical uctuation assay and the Lea-Coulson method (m/Nt where m is the number of resistant colonies and Nt is the total input).Supplementary Files

Figure 6 The
Figure 6

Figure 7 Characterization
Figure 7 Our data show that ItreS SM has approximately six-fold fewer colonies growing on antibiotics compared to wildtype.Assuming the same value of for both genotypes, this decrease could result from a six-fold lower persister-formation rate in ItreS SM while maintaining at the same level., which represents approximately a 20% decrease compared to its value in wildtype.This slight decrease alone cannot account for the six-fold change in , which must result from a sixfold decrease in the transition rate from the drug-sensitive to the drug-tolerant state.Overall, the uctuation assay data suggest that ItreS SM persisters are slightly more unstable and revert to being drug-sensitive more quickly.The observed difference in is primarily due to a six-fold lower rate of persister formation in ItreS SM compared to wildtype.
M. smegmatis wildtype, Flux RIF , or Flux INH in mid-log phase were incubated with 1X MIC RIF at 37°C.Bacteria were harvested at 0, 2, 4, 24, and 48 hrs and CFUs were determined by plating serial dilutions on m7H10.The cell-free supernatant was collected by ltering through a 0.22 µm lter.RIF was extracted by adding a precooled solution of LC-MS grade acetonitrile:methanl:H 2 O 2 (40:40:20) -40°C.RIF detection and quanti cation were performed using a Cogent Diamond Hydride Type C column (Microsolve Technologies) coupled with an Agilent Accurate Mass 6230 TOF and an Agilent 1290 Liquid